1. Field of the Invention
The present invention relates generally to herpes simplex virus (HSV)-based amplicon vectors. More specifically, the present invention relates to a herpes simplex virus (HSV)-based amplicon vector carrying a genomic DNA fragment. The invention also relates to methods of constructing a herpes simplex virus (HSV)-based amplicon. In one aspect of the invention, a method of converting any large capacity DNA cloning vector, such as, e.g. a bacterial artificial chromosome (BAC) or a P1-artificial chromosome (PAC) clone, into a herpes simplex virus (HSV) amplicon or hybrid HSV amplicon is provided. This method can be accomplished by using recombination, such as site-specific or homologous recombination, or ligation. According to this aspect of the invention, genomic DNA inserts within the large capacity DNA cloning vector can be delivered by infectious transfer to a target cell, and expressed, in vitro and in vivo. The present invention also relates to a system for the rapid creation of viral vectors carrying transgenes of interest. This aspect of the invention is accomplished through site-specific recombination between: (a) a large-capacity cloning vector carrying a viral genome, and (b) a transfer vector containing the transgene of interest. The present invention also relates to expression-ready genomic DNA libraries and methods of isolating a genomic DNA clone encoding a gene product with a preselected function.
2. Background Art
The terms “gene transfer” and “gene therapy” have been used to describe a variety of methods for delivering genetic material to a cell using viral or non-viral based vector systems. Substantial attention has been given to human gene therapy. The transfer of genetic material to a cell may one day become one of the most important forms of medicine. A variety of public and private institutions now participate in research and development related to the use of genetic material in therapeutic applications. Hundreds of human gene transfer protocols are being conducted at any given time with the approval of the Recombinant DNA Advisory Committee (RAC) and the National Institutes of Health (NIH). Most of these protocols focus on therapy, while others involve marking and non-therapeutic applications. The therapeutic protocols are primarily concerned with infectious diseases, monogenic diseases, and cancer. Gene-based therapies are now expanding into fields such as cardiovascular disease, autoimmune disease, and neurodegenerative disease. The availability of an efficient gene delivery and expression system is essential to the success and efficacy of gene-based therapy.
One method of delivering a gene of interest to a target cell is by using a viral-based vector. Techniques for the formation of vectors or virions are generally described in “Working Toward Human Gene Therapy,” Chapter 28 in Recombinant DNA, 2nd Ed, Watson, J. D. et al., eds., New York: Scientific American Books, pp. 567–581 (1992). An overview of viral vectors or virions that have been used in gene therapy can be found in Wilson, J. M., Clin. Exp. Immunol. 107(Suppl. 1):31–32 (1997), as well as Nakanishi, M. Crit. Rev. Therapeu. Drug Carrier Systems 12:263–310 (1995); Robbins, P. D., et al., Trends Biotechnol. 16:35–40 (1998); Zhang, J., et al., Cancer Metastasis Rev. 15:385–401 (1996); and Kramm, C. M., et al., Brain Pathology 5:345–381 (1995). Such vectors may be derived from viruses that contain RNA or DNA.
Specific examples of viral vector systems that have been utilized in the gene therapy art include: retroviruses (Vile, R. G., supra; U.S. Pat. Nos. 5,741,486 and 5,763,242); adenoviruses (Heise, C. et al., Nat. Med. 3:639–645 (1997)); adenoviral/retroviral chimeras (Bilbao, G., et al., FASEB J. 11:624–634 (1997); Feng, M., et al., Nat. Biotechnol. 15:866–870 (1997)); adeno-associated viruses (Flotte, T. R. and Carter, B. J., Gene Ther. 2:357–362 (1995); U.S. Pat. No. 5,756,283); herpes simplex virus I or II (Latchman, D. S., Mol. Biotechnol. 2:179–195 (1994); U.S. Pat. No. 5,763,217; Chase, M., et al., Nature Biotechnol. 16:444–448 (1998)); parvovirus (Shaughnessy, E., et al., Semin Oncol. 23:159–171 (1996)); and reticuloendotheliosis virus (Donburg, R., Gene Therap. 2:301–310 (1995)). Other viruses that can be used as vectors for gene delivery include poliovirus, papillomavirus, vaccinia virus, lentivirus, as well as hybrid or chimeric vectors incorporating favorable aspects of two or more viruses (Nakanishi, M. Crit. Rev. Therapeu. Drug Carrier Systems 12:263–310 (1995); Zhang, J., et al., Cancer Metastasis Rev. 15:385–401 (1996); Jacoby, D. R., et al., Gene Therapy 4:1281–1283 (1997)). Guidance in the construction of gene therapy vectors and the introduction thereof into affected animals for therapeutic purposes may be obtained in the above-referenced publications, as well as U.S. Pat. Nos. 5,631,236, 5,688,773, 5,691,177, 5,670,488, 5,601,818, and WO 95/06486.
The viral vectors mentioned above have advantages and disadvantages. For example, retroviruses have the ability to infect cells and have their genetic material integrated into the host cell with high efficiency. The development of a helper virus free packaging system for retrovirus vectors was a key innovation in the development of this vector system for human gene therapy. Retroviral helper virus free packaging systems generally employ the creation of a stable producer cell line that expresses a selected vector.
On a down side, however, numerous difficulties with retroviruses have been reported. For example, most retroviral vectors are not capable of gene transfer to postmitotic (nondividing) cells and are thus not applicable to the nervous system because most of the cells in the adult nervous system, especially neurons, are quiescent or postmitotic. In addition, outbreaks of wild-type virus from recombinant virus-producing cell lines have also been reported.
Difficulties have been noted with other viral vectors as well. Adenovirus vectors can only support limited long-term (2 months) gene expression, they appear to be gradually lost from neural cells, and moreover, they can cause both cytopathic effects and an immune response (Le Gal La Salle, G., et al., Science 259:988–990 (1993); Davidson et al., Nat. Genet. 3:219–223 (1993); Yang, Y., et al., J. Virol. 69:2004–2015 (1995)). Adeno-associated virus vectors cause minimal cytopathic effects and can support at least some gene expression for up to 4 months, but gene transfer is inefficient and these vectors can accept only ˜4 kb of foreign DNA (Kaplitt, M. G., et al., Nat. Genet. 8:148–154 (1994)).
The herpesviruses are a family of human viruses which include cytomegalovirus (CMV; 230 kb genome size), Epstein-Barr virus (EBV; 172 kb) and Herpes Simplex virus Types 1 and 2 (HSV-1 and 2; 152 kb). EBV and HSV-1 in particular have features which make them attractive as gene delivery vectors. EBV has a mechanism of prolonged extrachromosomal (episomal) maintenance in long-lived memory B-cells using the interaction of the latent origin of replication (oriP) and the viral protein EBV nuclear antigen-1 (EBNA-1) (Wolf, H., et al., Intervirology 35:26–39 (1993)). Plasmid vectors incorporating oriP and an expression cassette for EBNA-1 are maintained in certain cell types for prolonged periods (Yates, J. L., et al., Nature 313:812–815 (1985)).
Vectors based on herpes simplex virus (HSV), and especially HSV-1, have shown considerable promise as potent gene delivery vehicles for several reasons: the virus has a very large genome and thus can accommodate large amounts of foreign DNA (greater than 30 kb), the virus can persist long-term in cells (they establish latency), and can efficiently infect many different cell types, including post-mitotic neural cells (Breakefield, X. O., et al., “Herpes Simplex Virus Vectors for Tumor Therapy,” in The Internet Book of Gene Therapy: Cancer Gene Therapeutics, R. E. Sobol and K. J. Scanlon, eds. Appleton and Lange, Stamford, Conn., pp. 41–56 (1995); Glorioso, J. C., et al., “Herpes Simplex Virus as a Gene-Delivery Vector for the Central Nervous System,” in Viral Vectors: Gene Therapy and Neuroscience Applications, M. G. Kaplitt and A. D. Loewy, eds., Academic Press, New York, pp. 1–23 (1995)).
The recent manipulation of CMV, EBV and HSV-1 in bacteria (Messerle, M., et al., Proc. Natl. Acad. Sci. USA 94:14759–14763 (1997); Delecluse, H. J., et al., Proc. Natl. Acad. Sci. USA 95:8245–8250 (1998); Saeki, Y., et al., Hum. Gene Ther. 9:2787–2794 (1998)) is greatly assisting their progress as gene delivery vectors, and has led to the development of helper virus-free packaging systems for EBV and HSV-1 (Delecluse, H. J., et al., Proc. Natl. Acad. Sci. USA 95:8245–8250 (1998); Saeki, Y., et al., Hum. Gene Ther. 9:2787–2794 (1998)). Infectious amplicon vectors, which incorporate a viral origin of replication and a viral packaging signal into a bacterial plasmid, have been developed for both EBV and HSV-1.
HSV-1 amplicons carrying the oris replication origin and the pac signal have been widely used for gene delivery both in vivo and in vitro (Spaete, R. R., and Frenkel, N., Cell 30:295–304 (1982); Spaete, R. R., and Frenkel, N., Proc. Natl. Acad. Sci. USA 82:694–698 (1985); Geller, A. I., and Breakefield, X. O., Science 241:1667–1669 (1988); Sena-Esteves, M., et al., Mol. Ther. 2:9–15 (2000)). HSV amplicon vectors are one of the most versatile, most efficient, and least toxic, and have the largest transgene capacity of the currently available viral vectors. HSV-1 amplicon vectors can support some gene expression for up to one year (During, M. J., et al., Science 266:1399–1403 (1994)).
EBV amplicons carrying both the latent (oriP) and lytic (oriLyt) viral origins of replication together with the Terminal Repeats (TR) necessary for viral packaging have been used for gene transfer and expression in B-cell lines (Hammerschmidt, W., and Sugden, B., Cell 55:427–433 (1988); Hammerschmidt, W., and Sugden, B., Nature 340:393–397 (1989); Banerjee, S., et al., Nature Med. 1:1303–1308 (1995)). In addition, the large size of the herpesvirus genomes confers the potential for the delivery of very large transgenes. It is believed that the largest insert delivered by an HSV-1 amplicon previous to our study was 40 kb, and no expression from the insert was shown (Wang, X., et al., BioTechniques 27:102–106 (1999)).
The particular advantages of HSV-1 and EBV may be combined in a hybrid vector (Wang, S., et al., Gene Ther. 4:1132–1141 (1997)). HSV-1/EBV hybrid vectors packaged as HSV-1 amplicons are promising tools for gene delivery because: (i) HSV-1 has a high transgene capacity of approximately 150 kb; (ii) high-titre amplicon stocks can be produced by helper virus-free packaging systems; and (iii) the resulting virion particles have a broad cell tropism across a wide range of species. It is believed that HSV-1 is unique in being able to combine all these features. The addition of the EBV mechanism of episome retention allows long term persistence of the recircularized vector. The inclusion of a large genomic insert would further ensure such an episome can replicate in rodent cells (Wohlgemuth, J. G., et al., Gene Ther. 3:503–512 (1996)), further increasing the vector's utility in disease models.
Because HSV-1 encodes many toxic functions, improvements on the amplicon system have been targeted primarily at reducing the risk associated with the helper virus. First, replication-competent HSV-1, initially used as helper virus, was replaced by a temperature-sensitive (ts) mutant of HSV-1 (HSV-1 tsK; Preston, C., J. Virol. 29:257–284 (1979)). This mutant encodes a temperature-sensitive form of the essential HSV-1 infected cell protein (ICP) 4, allowing HSV-1 replication to proceed at 31° C., but not at 37° C. Amplicons packaged at 31 ° C. in the presence of HSV-1 tsK were successfully used to transfer the E. coli lacZ gene into primary cultures of rat neural cells (Geller, A. I. and Breakefield, X. O., Science 241:1667–1669 (1988)). Because the infection was performed at 37° C., the lytic cycle of the HSV-1 tsK helper virus present in the vector stock was blocked and cell damage was limited. Although replication of HSV-1 tsK was inhibited at the restrictive temperature, the expression of other viral genes caused cytopathic effects. Moreover, reversion to wild type (wt) HSV-1 occurred at a relatively high frequency.
To counter these problems, replication-defective mutants of HSV-1 were then used as helper viruses (Geller, A.I. et al., Proc. Natl. Acad. Sci. USA 87:8950–8954 (1990); Lim, F., et al., BioTechniques 20:458–469 (1996)). These mutants carry deletions in genes that are essential for virus replication, but they can support amplicon packaging in cells that complement the missing functions. In general, deletion-mutant packaging systems produce relatively high amplicon vector titers (106–107 transducing units per ml (t.u./ml)), a ratio of transducing vector units to helper virus of up to 1, and low levels of revertants with wt HSV-1 phenotype (<10−7 plaque forming units (PFU), per ml; Lim, F., et al., supra). However, many problems associated with the presence of helper virus in amplicon stocks still remained, including: (i) pronounced cytopathic effects and immune responses caused by gene expression from the helper virus; (ii) interactions between the helper virus and endogenous viruses; (iii) reversion of the helper virus to wt HSV-1; and (iv) disregulation of transgene expression by virus proteins.
Many of these problems have been overcome by the development of a packaging system for herpes virus vectors that was free of helper virus (Fraefel, C., et al., J. Virol. 70:7190–7197 (1996); International Patent Publication WO 97/05263, published February 13, 1997)). This system utilizes transient co-transfection of amplicon DNA with a set of five cosmids that overlap and represent the entire HSV-1 genome, but which are mutated to delete the DNA cleavage/packaging (pac) signals. Cunningham, C. and Davison, A. J., Virology 197:116–124 (1993), had demonstrated previously that after transfection into cells, an overlapping HSV-1 cosmid set can produce infectious virus progeny. By deleting the pac signals and making a pacminus helper virus genome, HSV-1 genomes that are potentially reconstituted from the cosmids via homologous recombination, are not packageable, but can still provide all the helper functions required for the replication and packaging of the co-transfected amplicon DNA. The resulting vector stocks are, therefore, virtually free of detectable helper virus and have titers of 106–107 t.u./ml of culture medium. Because of minimal sequence homology between the cosmids and the amplicon DNA (oris; 0.2–1 kb), the formation of a packageable and replication-competent HSV-1 genome is possible, but requires 6 recombination events, and is therefore very rare. Amplicon vector stocks, produced by using the helper virus-free packaging system, can efficiently transduce many different cell types, including neural cells and hepatocytes in culture and in vivo, while causing minimal to no cytopathic effects (Fraefel, C., et al., J. Virol. 70:7190–7197 (1996); Fraefel, C., et al., Mol. Med. 3:813–825 (1997); Fraefel, C., et al., “HSV-1 Amplicon” in Gene Therapy for Neurological Disorders and Brain Tumors, E. A. Chiocca and X. O. Breakefield, eds., Humana Press, Totowa, pp. 63–82 (1998); Johnston, K. M., et al., Hum. Gene Ther. 8:359–370 (1997); Aboody-Guterman, K. S., et al., NeuroReport 8:3801–3808 (1997)).
Even more recently, the helper virus-free herpes amplicon packaging system has been simplified further by reducing the number of clones representing the HSV-1 genome to a single clone (International Patent Publication WO 0034497; Saeki et al., Human Gene Therapy 9:2787–2794 (1998)). In this simplified system, a packaging vector comprising a single clone (i.e., a BAC containing the entire HSV-1 genome) was used as “helper virus.”
Most current viral vectors have a transgene capacity limited to the delivery of cDNA-based expression cassettes, often driven by strong heterologous viral promoters. In contrast, the delivery of a genomic DNA transgene driven by the native promoter, flanked by the regulatory regions and including introns, offers the potential for investigating and exploiting the physiological control of gene expression (Li, Q., et al., Trends Genet. 15:403–408 (1999); Blackwood, E. M. and Kadonaga, J. T., Science 281:60–63 (1998)). Many studies have demonstrated the advantages of using genomic DNA in cell culture and transgenic animal models (Yang, X. W., et al., Nature Biotechnol. 15:859–865 (1997); Wade-Martins, R., et al., Nature Biotech 18:1311–1314 (December 2000); Schiedner, G., et al., Nature Genet. 18:180–183 (1998); Peterson, K. R., et al., Proc. Natl. Acad. Sci. USA 90:11207–11211 (1993)). Viral vectors are an efficient means of delivering genes to cells, but the size of most genomic loci precludes their use in current viral systems.
The development of bacterial artificial chromosomes (BACs) (Shizuya, H., et al., Proc. Natl. Acad. Sci. USA 89:8794–8797 (1992)) and P1-artificial chromosomes (PACs)(Ioannou, P. A., et al., Nature Genet. 6:84–89 (1994)) has greatly aided physical mapping projects and genomic sequencing. BACs and PACs have many advantages over yeast artificial chromosomes (YACs) for cloning large DNA inserts (Monaco, A. P., and Larin, Z., Trends Biotech. 12:280–286 (1994)), including the ease of preparation of microgram quantities of vector. Nonetheless, the use of all three vectors in gene expression studies is restricted by the difficulty of transferring and retaining intact pieces of genomic DNA >100 kb in human cells. As a result of the human genome sequencing projects, virtually the entire human genome is now covered by BAC contigs, which makes BACs an excellent platform for functional genomics studies (Simon, M. I., Nature Biotechnol. 15:839 (1997)).
Gene expression from BACs and PACs has been demonstrated in cell culture systems (Wade-Martins, R., et al., Nature Biotech 18:1311–1314 (December 2000); Compton, S. H., et al., Gene Ther. 7:1600–1605 (2000); Kim, S. Y., et al., Genome Res. 8:404–412 (1998)) and in transgenic animal models (Antoch, M. P., et al., Cell 89:655–667 (1997); Yang, X. W., et al., Nature Genet. 22:327–335 (1999)). Wade-Martins et al. has developed a large insert shuttle vector for gene expression in human cells based on a fusion of the BAC and EBV episome technologies (Wade-Martins, R., et al., Nature Biotech 18:1311–1314 (December 2000); Wade-Martins, R., et al., Nucleic Acids Res. 27:1674–1682 (1999)). The vector was used for complementation of a cell culture phenotype by a genomic DNA transgene retained in human cells as an EBV-based episome (Wade-Martins, R., et al., Nature Biotech 18:1311–1314 (December 2000)). Extrachromosomal maintenance of the construct prevented DNA rearrangement often seen on construct integration. The vector described by Wade-Martins, supra, is based solely on EBV features, but not HSV-1. Moreover, it is not an infectious viral system. It can only be transferred to mammalian cells by physical transfection, which is much less efficient than viral transfer. Even if the Wade-Martins vector were to be turned into an infectious EBV-based vector, it would be severely limited by the problems of an EBV system, namely (i) the inability to make high titre virus and (ii) a very narrow range of cell infectivity.
Westphal, E. M., et al., Human Gene Therapy 9:1863–1873 (September 1998) and international Patent Publication WO 00/12693 to Vos et al. relate to a vector system for shuttling large genomic inserts from preexisting BAC or PAC libraries into human cells. The system utilizes a hybrid BAC-HAEC (human artificial episomal chromosome), which contains an F-based replication system as in BAC and the EBV oriP, for replication in human cells. Transcription of the human beta-globin gene (185 kb) was observed in vitro.
U.S. Pat. No. 6,143,566 to Heintz et al. relates to targeted BAC modification. This patent teaches a method for directly modifying an independent origin based cloning vector (such as a BAC, in one specific embodiment) in recombination deficient host cells, including generating deletions, substitutions, and/or point mutations in a specific gene contained in the cloning vector. The modified cloning vector may be used to introduce a modified heterologous gene into a host cell. In one Example presented, a modified BAC was inserted into a murine subject animal, and in vivo heterologous gene expression demonstrated. The methodology of this invention involves homologous recombination of the cloning vector with a conditional replication shuttle vector in a RecA.sup.—host cell, wherein the conditional replication shuttle vector encodes a RecA-like protein. In a preferred embodiment, the vector is a BAC that has undergone homologous recombination with the temperature sensitive shuttle vector pSV1.RecA.
Clearly, there is a need in the art to simplify and enhance viral gene delivery systems for large capacity DNA cloning vectors, such as, e.g., BACs and PACs, so that genomic DNA inserts (and in particular large genomic DNA inserts) within the large capacity DNA cloning vector can be more easily transferred by infection into cells (in vitro and in vivo), in order to study and exploit gene expression and function. Since, as a result of the efforts of the Human Genome Project, the human genome is now covered by BAC contigs (i.e., BACs comprising overlapping genomic fragments), this vector system has utility for the efficient delivery of large genomic transgenes in functional genomics studies and gene therapy applications.